Microvesicle Fraction Research Articles

Discovery of Cell Number-Interstitial Fluid Volume (CIF) Ratio Reveals Secretory Autophagy Pathway to Supply eHsp90α for Wound Healing.

Cell secretion repairs tissue damage and restores homeostasis throughout adult life. The extracellular heat shock protein-90alpha (eHsp90α) has been reported as an exosome cargo and a potential driver of wound healing. However, neither the mechanism of secretion nor the genetic evidence for eHsp90α in wound healing has been substantiated. Herein, we show that tissue injury causes massive deposition of eHsp90α in tissues and secretion of eHsp90α by cells. Sequential centrifugations of conditioned medium from relevant cell lines revealed the relative distributions of eHsp90α in microvesicle, exosome and trypsin-sensitive supernatant fractions to be approximately <2%, <4% and >95%, respectively. Establishing the cell-number-to-interstitial-fluid-volume (CIF) ratio for the microenvironment of human tissues as 1 × 109 cells: 1 mL interstitial fluid enabled us to predict the corresponding tissue concentrations of eHsp90α in these fractions as 3.74 μg/mL, 5.61 μg/mL and 178 μg/mL. Remarkably, the 178 μg/mL eHsp90α matches the previously reported 100-300 μg/mL of recombinant eHsp90α whose topical application promotes maximum wound healing in animal models. More importantly, we demonstrate that two parallel secretory autophagy-regulating gene families, the autophagy-regulating (AR) genes and the Golgi reassembly-stacking protein (GRASP) genes work together to mediate the secretion of the physiological concentration of eHsp90α to promote wound healing. Thus, utilization of the CIF ratio-based extrapolation method may enable investigators to rapidly predict biomarker targets from cell-conditioned-medium data.

Abstract A048: Utero-tubal lavage proteomic analysis: detection of ovarian cancer in BRCA mutation carriers

Abstract A048: Utero-tubal lavage proteomic analysis: detection of ovarian cancer in BRCA mutation carriers

Proteomic signature for detection of high-grade ovarian cancer in germline BRCA mutation carriers.

No current screening methods for high-grade ovarian cancer (HGOC) guarantee effective early detection for high-risk women such as germline BRCA mutation carriers. Therefore, the standard-of-care remains risk-reducing salpingo-oophorectomy (RRSO) around age 40. Proximal liquid biopsy is a promising source of biomarkers, but sensitivity has not yet qualified for clinical implementation. We aimed to develop a proteomic assay based on proximal liquid biopsy, as a decision support tool for monitoring high-risk population. Ninety Israeli BRCA1 or BRCA2 mutation carriers were included in the training set (17 HGOC patients and 73 asymptomatic women), (BEDOCA trial; ClinicalTrials.gov Identifier: NCT03150121). The proteome of the microvesicle fraction of the samples was profiled by mass spectrometry and a classifier was developed using logistic regression. An independent cohort of 98 BRCA mutation carriers was used for validation. Safety information was collected for all women who opted for uterine lavage in a clinic setting. We present a 7-protein diagnostic signature, with AUC >0.97 and a negative predictive value (NPV) of 100% for detecting HGOC. The AUC of the biomarker in the independent validation set was >0.94 and the NPV >99%. The sampling procedure was clinically acceptable, with favorable pain scores and safety. We conclude that the acquisition of Müllerian tract proximal liquid biopsies in women at high-risk for HGOC and the application of the BRCA-specific diagnostic assay demonstrates high sensitivity, specificity, technical feasibility and safety. Similar classifier for an average-risk population is warranted.

Abstract P252: Differential Expression Of ACE2, Neprilysin And ACE In Extracellular Vesicles Isolated From Human Urine

The Ang II convertase ACE2 is present in urine and may correlate with various renal pathologies. We reported functional ACE2 in urinary exosomes and now extend our studies to distinguish exosomes (EXOs) and microvesicles (MVs) containing ACE2, as well as neprilysin (NEP) and ACE. EXO purification was modified by an intermediate centrifugation step (20k xg, 60 mins) to pellet MVs followed by 100k xg to pellet EXOs. The MV, EXO, and 100k supernatant (soluble, SOL) fractions were assessed for ACE2 activity with MCA-APK-DNP, and NEP and ACE activities with MCA-RPPGFSAFK-Dnp with selective inhibitors. Peptidase activities were expressed as relative fluorescent units (RFU x10 6 /min/mL), and data are means ± SEM (*P&lt;0.05; **P&lt;0.01, N=4). Morning collections from male subjects [51 to 65 years of age, non-smokers] were collected, treated with Cibacron blue to bind albumin, 0.2 μM filtration, and 100 kDa Amicon concentration prior to high speed and ultracentrifugation steps. ACE2 activity was &gt;3-fold higher in the EXO versus the MV fractions with minor activity in the SOL fraction. In contrast, NEP activity was highest in the MV fraction [~2-fold &gt; EXO] while endopeptidase activity was barely detectable in the SOL fraction. ACE activity was similar in MVs and EXOs, but NEP activity was 40-fold higher than ACE in both the MV and EXO fractions. Differential centrifugation of the 100-kDa urine concentrate reveals a unique profile of ACE2, NEP and ACE expression in MVs and EXOs. The functional significance of these peptidase activities in different vesicular populations is currently unknown; however, their origin may reflect the distinct regulation and release of ACE2 and NEP/ACE in urinary vesicles.

Human mesenchymal stromal cells release functional mitochondria in extracellular vesicles.

Cartilage and other skeletal soft tissues heal poorly after injury, in part due to their lack of vascularity and low metabolic rate. No pharmacologic approaches have proven effective in preventing chronic degenerative disease after joint injury. Mesenchymal stromal cells (MSCs) have been investigated for their ability to treat pain associated with osteoarthritis (OA) and preserve articular cartilage. Limitations of MSCs include variability in cell phenotype, low engraftment and retention rates, and inconsistent clinical outcomes. Therefore, acellular biologic therapies such as extracellular vesicles (EVs) are currently being investigated. MSC-derived EVs have been found to replicate many of the therapeutic effects of their cells of origin, but the mechanisms driving this remain unclear. Recent evidence in non-orthopedic tissues suggests MSCs can rescue injured cells by donating mitochondria, restoring mitochondrial function in recipient cells, preserving cell viability, and promoting tissue repair. Our group hypothesized that MSCs package mitochondria for export into EVs, and that these so-called “mitoEVs” could provide a delivery strategy for cell-free mitochondria-targeted therapy. Therefore, the goals of this study were to: 1) characterize the vesicle fractions of the MSCs secretome with respect to mitochondrial cargoes, 2) determine if MSC-EVs contain functional mitochondria, and 3) determine if chondrocytes can take up MSC-derived mitoEVs. We isolated exosome, microvesicle, and vesicle-free fractions from MSC-conditioned media. Using a combination of dynamic light scattering and nanoparticle tracking, we determined that MSC-EV populations fall within the three size categories typically used to classify EVs (exosomes, microvesicles, apoptotic bodies). Fluorescent nanoparticle tracking, immunoblotting, and flow cytometry revealed that mitochondrial cargoes are abundant across all EV size populations, and mitoEVs are nearly ubiquitous among the largest EVs. Polarization staining indicated a subset of mitoEVs contain functional mitochondria. Finally, flow cytometry and fluorescent imaging confirmed uptake of mitoEVs by chondrocytes undergoing rotenone/antimycin-induced mitochondrial dysfunction. These data indicate that MSCs package intact, functional mitochondria into EVs, which can be transferred to chondrocytes in the absence of direct cell-cell interactions. This work suggests intercellular transfer of healthy MT to chondrocytes could represent a new, acellular approach to augment mitochondrial content and function in poorly-healing avascular skeletal soft tissues.

AQP4 labels a subpopulation of white matter-dependent glial radial cells affected by pediatric hydrocephalus, and its expression increased in glial microvesicles released to the cerebrospinal fluid in obstructive hydrocephalus

Hydrocephalus is a distension of the ventricular system associated with ventricular zone disruption, reactive astrogliosis, periventricular white matter ischemia, axonal impairment, and corpus callosum alterations. The condition's etiology is typically attributed to a malfunction in classical cerebrospinal fluid (CSF) bulk flow; however, this approach does not consider the unique physiology of CSF in fetal and perinatal patients. The parenchymal fluid contributes to the glymphatic system, and plays a fundamental role in pediatric hydrocephalus, with aquaporin 4 (AQP4) as the primary facilitator of these fluid movements. Despite the importance of AQP4 in the pathophysiology of hydrocephalus, it’s expression in human fetal life is not well-studied. This manuscript systematically defines the brain expression of AQP4 in human brain development under control (n = 13) and hydrocephalic conditions (n = 3). Brains from 8 postconceptional weeks (PCW) onward and perinatal CSF from control (n = 2), obstructive (n = 6) and communicating (n = 6) hydrocephalic samples were analyzed through immunohistochemistry, immunofluorescence, western blot, and flow cytometry. Our results indicate that AQP4 expression is observed first in the archicortex, followed by the ganglionic eminences and then the neocortex. In the neocortex, it is initially at the perisylvian regions, and lastly at the occipital and prefrontal zones. Characteristic astrocyte end-feet labeling surrounding the vascular system was not established until 25 PCW. We also found AQP4 expression in a subpopulation of glial radial cells with processes that do not progress radially but, rather, curve following white matter tracts (corpus callosum and fornix), which were considered as glial stem cells (GSC). Under hydrocephalic conditions, GSC adjacent to characteristic ventricular zone disruption showed signs of early differentiation into astrocytes which may affect normal gliogenesis and contribute to the white matter dysgenesis. Finally, we found that AQP4 is expressed in the microvesicle fraction (p < 0.01) of CSF from patients with obstructive hydrocephalus. These findings suggest the potential use of AQP4 as a diagnostic and prognostic marker of pediatric hydrocephalus and as gliogenesis biomarker.

P2X7 promotes metastatic spreading and triggers release of miRNA-containing exosomes and microvesicles from melanoma cells

Tumor growth and metastatic spreading are heavily affected by the P2X7 receptor as well as microvesicles and exosomes release into the tumor microenvironment. P2X7 receptor stimulation is known to trigger vesicular release from immune and central nervous system cells. However, P2X7 role in microvesicles and exosomes delivery from tumor cells was never analyzed in depth. Here we show that P2X7 is overexpressed in patients affected by metastatic malignant melanoma and that its expression closely correlates with reduced overall survival. Antagonism of melanoma cell-expressed P2X7 receptor inhibited in vitro anchorage-independent growth and migration and in vivo dissemination and lung metastasis formation. P2X7 stimulation triggered the release of miRNA-containing microvesicles and exosomes from melanoma cells, profoundly altering the nature of their miRNA content, as well as their dimensions and quantity. Among the more than 200 miRNAs that we found up-or-down-modulated for each vesicular fraction tested, we identified three miRNAs, miR-495-3p, miR-376c-3p, and miR-6730-3p, that were enriched in both the exosome and microvesicle fraction in a P2X7-dependent fashion. Interestingly, upon transfection, these miRNAs promoted melanoma cell growth or migration, and their vesicular release was minimized by P2X7 antagonism. Our data unveil an exosome/microvesicle and miRNA-dependent mechanism for the pro-metastatic activity of the P2X7 receptor and highlight this receptor as a suitable prognostic biomarker and therapeutic target in malignant melanoma.

Breast Cancer-Derived Microvesicles Are the Source of Functional Metabolic Enzymes as Potential Targets for Cancer Therapy.

Membrane-derived extracellular vesicles, referred to as microvesicles (MVs), have been proposed to participate in several cancer diseases. In this study, MV fractions were isolated by differential ultracentrifugation from a metastatic breast cancer (BC) cell line MDA-MB-231 and a non-cancerous breast cell line MCF10A, then analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. A total of 1519 MV proteins were identified from both cell lines. The data obtained were compared to previously analyzed proteins from small extracellular vesicles (sEVs), revealing 1272 proteins present in both MVs and sEVs derived from the MDA-MB-231 cell line. Among the 89 proteins unique to MDA-MB-231 MVs, three enzymes: ornithine aminotransferase (OAT), transaldolase (TALDO1) and bleomycin hydrolase (BLMH) were previously proposed as cancer therapy targets. These proteins were enzymatically validated in cells, sEVs, and MVs derived from both cell lines. The specific activity of OAT and TALDO1 was significantly higher in MDA-MB-231-derived MVs than in MCF10A MVs. BLMH was highly expressed in MDA-MB-231-derived MVs, compared to MCF10A MVs. This study shows that MVs carry functional metabolic enzymes and provides a framework for future studies of their biological role in BC and potential in therapeutic applications.

Cutaneous T-Cell Lymphoma (CTCL) Cell Line-Derived Extracellular Vesicles Contain HERV-W-Encoded Fusogenic Syncytin-1

Cutaneous T-Cell Lymphoma (CTCL) Cell Line-Derived Extracellular Vesicles Contain HERV-W-Encoded Fusogenic Syncytin-1

Characterization of single microvesicles in plasma from glioblastoma patients.

Extracellular vesicles (EV) are shed by tumor cells but little is known about their individual molecular phenotypes and heterogeneity. While exosomes have received considerable attention, much less is known about larger microvesicles. Here we profile single microvesicles (MV) and exosomes from glioblastoma (GB) cells and MV from the plasma of patients. EV secreted from mouse glioma GL261 and human primary GBM8 cell lines as well as from the plasma of 8 patients with diagnoses of GB and 2 healthy controls were isolated and processed for single vesicle analysis. EV were immobilized on glass slides and the heterogeneity of vesicle and tumor markers were analyzed at the single vesicle level. We show that (i) MV are abundant, (ii) only a minority of MV expresses putative MV markers, and (iii) MV share tetraspanin biomarkers previously thought to be diagnostic of exosomes. Using MV capture and staining techniques that allow differentiation of host cell and GB-derived MV we further demonstrate that (i) tumoral MV often present as <10% of all MV in GB patient plasma, and (ii) there is extensive heterogeneity in tumor marker expression in these tumor-derived MV. These results indicate that single MV analysis is likely necessary to identify rare tumoral MV populations and the single vesicle analytical technique used here can be applied to both MV and exosome fractions without the need for their separation from each other. These studies form the basis for using single EV analyses for cancer diagnostics.

Diurnal variation of circulating microvesicles is associated with the severity of obstructive sleep apnoea.

Microvesicles (MVs) have been implicated in the pathomechanism of obstructive sleep apnoea (OSA); however, the results are inconsistent, possibly due to an unrevealed temporal variation in circulating MV levels. We aimed to investigate the diurnal changes of MV fractions in OSA. Peripheral blood was taken from 18 patients with OSA and 9 healthy subjects at different time points (11:00, 17:00, 21:00, 01:30 and 06:00). Samplings were repeated in nine OSA patients after 2months of continuous positive airway pressure (CPAP) therapy. CD41+, CD62P+, glycophorin A+ and Annexin V+ MVs were determined with flow cytometry. Areas under the MV concentrations-time curves (AUC) were calculated and correlated with the severity of OSA. A significant diurnal variability of plasma CD41+ and Annexin V+ MVs was observed only in OSA with a marked peak at 17:00. There was a direct correlation between CD41+ MV AUCs and the severity of OSA. CPAP treatment reduced diurnal variability in both CD41+ and Annexin V+ MV levels. The relationship between the diurnal variability of CD41+ MVs and disease severity as well as the effect of CPAP treatment on MV levels support the role of MVs in the pathophysiology of OSA. More importantly, considering the significant diurnal variation in circulating MV levels, introduction of strict protocols for blood sampling is required for MV measurements.

Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis

The human placenta releases multiple types and sizes of syncytiotrophoblast (STB) extracellular vesicles (EV) into the maternal circulation that exhibit diverse biological activities. The placental perfusion technique enables isolation of these STBEV, but conventional flow cytometry can only be used to phenotype EV down to ∼300nm in size. Fluorescence Nanoparticle Tracking Analysis (fl-NTA) has the potential to phenotype EV down to ∼50nm, thereby improving current characterisation techniques. The aims of this study were to prepare microvesicle and exosome enriched fractions from human placental perfusate (n=8) and improve fl-NTA STBEV detection. Differential centrifugation and filtration effectively removed contaminating red blood cells from fresh placental perfusates and pelleted a STB microvesicle (STBMV) fraction (10,000×g pellet – 10KP; NTA modal size 395±12nm), enriched for the STB marker placental alkaline phosphatase (PLAP) and a STB exosome (STBEX) fraction (150,000×g pellet – 150KP; NTA modal size 147±6nm), enriched for PLAP and exosome markers Alix and CD63. The PLAP positivity of ‘standard’ 10KP and 150KP pools (four samples/pool), determined by immunobead depletion, was used to optimise fl-NTA camera settings. Individual 10KP and 150KP samples (n=8) were 54.5±5.7% (range 17.8–66.9%) and 30.6±5.6% (range 3.3–51.7%) PLAP positive, respectively. We have developed a reliable method for enriching STBMV and STBEX from placental perfusate. We also standardised fl-NTA settings and improved measurement of PLAP positive EV in STBMV. However, fl-NTA is not as sensitive as anti-PLAP Dynabead capture for STBEX detection, possibly due to STBEX having lower surface expression of PLAP. These important developments will facilitate more detailed studies of the role of STBMV and STBEX in normal and pathological pregnancies.

Supernatants from stored red blood cell (RBC) units, but not RBC-derived microvesicles, suppress monocyte function in vitro.

We have previously shown that critically ill children transfused with red blood cells (RBCs) of longer storage durations have more suppressed monocyte function after transfusion compared to children transfused with fresher RBCs and that older stored RBCs directly suppress monocyte function in vitro, through unknown mechanisms. We hypothesized that RBC-derived microvesicles (MVs) were responsible for monocyte suppression. To determine the role of stored RBC unit-derived MVs, we cocultured monocytes with supernatants, isolated MVs, or supernatants that had been depleted of MVs from prestorage leukoreduced RBCs that had been stored for either 7 or 30 days. Isolated MVs were characterized by electron microscopy and flow cytometry. Monocyte function after coculture experiments was measured by cytokine production after stimulation with lipopolysaccharide (LPS). Monocyte function was suppressed after exposure to supernatants from 30-day RBC units compared to monocytes cultured in medium alone (LPS-induced tumor necrosis factor-α production, 17,611 ± 3,426 vs. 37,486 ± 5,598 pg/mL; p = 0.02). Monocyte function was not suppressed after exposure to MV fractions. RBC supernatants that had been depleted of MVs remained immunosuppressive. Treating RBC supernatants with heat followed by RNase (to degrade protein-bound RNA) prevented RBC supernatant-induced monocyte suppression. Our findings implicate soluble mediators of stored RBC-induced monocyte suppression outside of MV fractions and suggest that extracellular protein-bound RNAs (such as microRNA) may play a role in transfusion-related immunomodulation.

IDO is highly expressed in breast cancer and breast cancer-derived circulating microvesicles and associated to aggressive types of tumors by in silico analysis

Indoleamine-2,3-dioxygenase (IDO) has been established as a normal mechanism of peripheral tolerance and immunosuppression. Besides, malignant tumors release microvesicles (MV) related with tumor dissemination. The aims of this study were to determine the expression of IDO in breast cancer and circulating microvesicles from breast cancer patients and to perform an in silico analysis to find genes co-expressed to IDO. One hundred and twenty-two tissue and serum breast samples (91 malignant, 21 benign, and 10 normal), and MCF7, MDA-MB-231, and T47D breast cancer cell lines were included. Standard immunohistochemistry (IHC), immunocytochemistry (ICC), Western blot (WB), and RT-PCR were employed. Microvesicle isolation from plasma samples was obtained by serial centrifugation and ultracentrifugation. By IHC, 60 % breast cancer, 43 % benign, and 20 % normal samples were positive. Significant differences were found among normal, benign, and malignant samples. Breast cancer stages I, II, and III expressed IDO in 42, 66, and 71 % of samples, respectively, while breast cancer cell lines also reacted; by WB, 9/25 microvesicles fractions showed bands at 42 kD. In silico analysis of IDO 1 gene expression in breast cancer showed its association with several genes related to immune response and apoptosis. Moreover, IDO and co-expressed genes were found predominately in basal and erbB2 subtypes. The cumulative data indicate a high expression of IDO in breast cancer which increased with higher stages. Furthermore, IDO was found in association with circulating breast cancer MV, while experimental and in silico gene expression revealed that IDO was mainly expressed in a triple-negative subgroup.

Abstract LB-108: Exosome profiling of mRNAs and miRNAs in serum samples from GBM patients .

Abstract Background: Biomarker discovery and development in oncology has traditionally required the use of tissue, either FFPE or frozen. In recent years, the use of Circulating Tumor Cells (CTCs), cell free DNA (cfDNA), and most recently, exosomes, has allowed biomarker research in biofluids to evolve. Exosomes contain mRNA, microRNA, DNA and protein in a protected, highly stable vesicle. Recently, we have shown that large panels of mRNA genes can be run on small samples of frozen, archived serum. The ability to assess the expressions patterns in blood represents a potentially important development for biomarker research and the development of non-invasive diagnostics. Methods: Frozen, archived serum (2ml) was obtained from a glioblastoma clinical trial cohort, containing serum from both responders and non-responders to a novel therapy. We isolated the microvesicle fraction from the serum, extracted the RNA, and qualified the RNA using an Agilent 2100 Bioanalyzer on an Agilent 6000 RNA Picochip. A standard in-house control extraction was processed in parallel and subjected to quality control on the Bioanalyzer as well as RT-qPCR. The first cohort of samples (n = 60) were reverse transcribed to cDNA, and the cDNA was assayed on a human cancer mRNA panel Array by qPCR (Life Technologies).The cancer panel includes a total of 648 cancer related genes including endogenous controls. The second cohort of samples (n = 40) was reverse transcribed and analyzed by a miRNA panel of 758 different miRNA assays and controls. Results: Our data clearly demonstrate the ability to detect relevant gene expression patterns in microvesicle RNA isolated from patient serum. High quality RNA was extracted from the samples received, and an average of 80% of the 648 cancer associated mRNA genes and 36% of the miRNAs were detected in the samples with the volumes used in this study. Data was demonstrated to be reproducible between and among samples and suggests a biological pattern of RNAs in serum exosomes that could be used to monitor response to treatment. Conclusions: RNA isolated from the microvesicle fraction of serum contains abundant, high-quality RNA, including mRNA. The analysis of a large panel of genes from a small sample of serum is possible, demonstrating that RNA isolated from microvesicles from peripheral blood has the potential to be used as a source of genetic material for non-invasive diagnostic tests. Citation Format: Johan Skog, Stefan Bentink, Lori LoGuidice, Charlotte Romain, Susan Belzer, Mikkel Noerholm. Exosome profiling of mRNAs and miRNAs in serum samples from GBM patients . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-108. doi:10.1158/1538-7445.AM2013-LB-108

Alteration in Abundance and Compartmentalization of Inflammation-Related miRNAs in Plasma After Intracerebral Hemorrhage

We tested the hypothesis that circulating microRNAs (miRNAs) present in plasma might display a specific signature in patients with intracerebral hemorrhage. Global miRNA profiles were determined with the Agilent Human miRNA Microarray platform, and validated by quantitative polymerase chain reaction. A subset of 30 miRNAs were selectively upregulated in both male and female patients with intracerebral hemorrhage. Network analysis revealed that the most significantly overrepresented biological process associated with these miRNAs was inflammation. Unlike the plasma miRNAs in healthy controls, which were detected exclusively in the microvesicle fraction, the deregulated plasma miRNAs after intracerebral hemorrhage were present in both the microvesicle and the supernatant fractions. Intracerebral hemorrhage altered both the abundance and the compartmentalization of several inflammation-related miRNAs in plasma.

Immune modulation of T-cell and NK (natural killer) cell activities by TEXs (tumour-derived exosomes)

Body fluids of cancer patients contain TEXs (tumour-derived exosomes). Tumours release large quantities of TEXs, and the protein content of exosome or MV (microvesicle) fractions isolated from patients' sera is high. TEXs down-regulate functions of immune cells, thus promoting tumour progression. We isolated TEXs from tumour cell supernatants and sera of patients with solid tumours or AML (acute myelogenous leukaemia). The molecular profile of TEXs was distinct from that of circulating exosomes derived from normal cells. TEXs were co-incubated with activated T-cells, conventional CD4(+) CD25(neg) T-cells or CD56(+) CD16(+) NK (natural killer) cells respectively. TEXs down-regulated CD3ζ and JAK3 (Janus kinase 3) expression in primary activated T-cells and mediated Fas/FasL (Fas ligand)-driven apoptosis of CD8(+) T-cells. TEXs promoted CD4(+) CD25(neg) T-cell proliferation and their conversion into CD4(+) CD25(hi)FOXP3+ (FOXP3 is forkhead box P3) Treg cells (regulatory T-cells), which also expressed IL-10 (interleukin 10), TGFβ1 (transforming growth factor β1), CTLA-4 (cytotoxic T-lymphocyte antigen 4), GrB (granzyme B)/perforin and effectively mediated suppression. Neutralizing antibodies specific for TGFβ1 and/or IL-10 inhibited the ability of TEXs to expand Treg cells. TEXs obtained at diagnosis from AML patients' sera were positive for blast-associated markers CD33, CD34, CD117 and TGFβ1, and they decreased cytotoxic activity of NK cells isolated from NC (normal control) donors, induced Smad phosphorylation and down-regulated NKG2D receptor expression. Correlations between the TEX molecular profile or TEX protein levels and clinical data in cancer patients suggest that TEX-mediated effects on immune cells are prognostically important. In contrast with exosomes released by normal cells, TEXs have immunosuppressive properties and are involved in regulating peripheral tolerance in patients with cancer.

Copper modulates the large dense core vesicle secretory pathway in PC12 cells

Copper (Cu) is an essential biometal involved in a number of cell functions. Abnormal Cu homeostasis has been identified as a major factor in a number of neurodegenerative disorders. However, little is known about how cells of brain origin maintain Cu homeostasis and in particular, how they respond to an elevated Cu environment. Understanding these processes is essential to obtaining a greater insight into the pathological changes in neurodegeneration and ageing. Although previous studies have shown that Cu in neurons can be associated with synaptic function, there is little understanding of how Cu modulates the regulated secretory vesicle pathways in these cells. In this study, we examined the effect of elevated intracellular Cu on proteins associated with the regulated secretory vesicle pathway in NGF-differentiated PC12 cells that exhibit neuronal-like properties. Increasing intracellular Cu with a cell-permeable Cu-complex (Cu(II)(gtsm)) resulted in increased expression of synaptophysin and robust translocation of this and additional vesicular proteins from synaptic-like microvesicle (SLMV) fractions to chromogranin-containing putative large dense core vesicle (LDCV) fractions in density gradient preparations. The LDCV fractions also contained substantially elevated Cu levels upon treatment of cells with Cu(II)(gtsm). Expression of the H(+) pump, V-ATPase, which is essential for vesicle maturation, was increased in Cu-treated cells while inhibition of V-ATPase prevented translocation of synaptophysin to LDCV fractions. Cu treatment was found to inhibit release of LDCVs in chromaffin cells due to reduced Ca(2+)-mediated vesicle exocytosis. Our findings demonstrate that elevated Cu can modulate LDCV metabolism potentially resulting in sequestration of Cu in this vesicle pool.

Human ether-a-go-go-related gene K+ channels regulate shedding of leukemia cell-derived microvesicles

Microvesicles (MVs) are released by various cancer cells, including leukemia cells. They can “hijack” membrane components from their parental cells and exert pleiotropic effects on tumor progression. Human ether-a-go-go-related gene (hERG1) K + channels are highly expressed in cancer cells and appear of exceptional importance in favoring cancer development. Given the attributes of MVs and hERG1 K + channels in disease progression, we investigated the putative relationship between hERG1 K + channels and MVs in leukemia. The protein content of MVs isolated from K562 cell supernatants was significantly higher than that from HL-60 cells. The molecular profile of these MVs showed that in addition to the myeloid lineage antigen (CD11b), MVs contained hERG1 K + channels. Interestingly, inhibition of hERG1 K + channels rapidly reduced MV fractions in supernatants. Furthermore, MVs created positive feedback loops to facilitate leukemogenesis. Upon exposure to MVs, the plasma membrane expression of hERG1 protein was in turn up-regulated, the migration of leukemia cells was significantly increased, and the adhesion of leukemia cells to human umbilical vein endothelial cells (HUVECs) was markedly enhanced. Importantly, hERG1 K + channel inhibitor E-4031 impaired these effects. We conclude that leukemia cell-derived MVs can “hijack” the plasma membrane hERG1 K + channels, which regulate the release of MVs and their biological effects upon leukemia cells.

AML-Derived Microvesicles as Mediators of Cell-Cell Signaling In the Leukemic Microenvironment

AbstractAbstract 1700Directed signaling between acute myelogenous leukemia (AML) cells and the bone marrow microenvironment plays an important role in leukemia cell survival. How leukemic blasts transform the homeostatic bone marrow microenvironment is not fully explained by known ligand-receptor interactions. Microvesicles (MV) are membrane-enclosed compartments that, depending on cell specific pathways, range in diameter from 50–100 nm (exosomes) to 100–1000 nm (microparticles) and are constitutively released into circulation in many tissues. Biogenesis has been found to be upregulated in a range of cancers. We hypothesized that MV-mediated transfer of signaling components by AML blasts may contribute to intercellular signaling in the leukemic niche. In an initial screen, we characterized MV that we recovered from the culture supernatant of multiple AML-derived cell lines, as well as AML cells from patients. Live-cell imaging of primary AML blasts using the exosomal pathway-specific dye N-Rh-PE revealed that at least a portion of these vesicles could be categorized as exosomes. Consistent with the non-random incorporation of molecular cargo observed in recent studies with solid tumors, we found the protein and RNA content of MV to be distinct from cellular lysates. In a focused, candidate target approach, our experiments showed that MV contain mRNA transcripts encoding the broadly relevant AML prognostic markers FLT3-ITD, NPM1, and IGF-1R. In both cell lines and cultured primary AML cells, insulin receptor (IR) and IGF-1R mRNAs were enriched up to ∼400-fold over GAPDH in MV versus cellular fractions. The IGF-1R receptor tyrosine kinase (RTK) transduces growth and survival signals, and the IGF-I signaling pathway has recently been identified as a potentially important candidate for molecularly targeted drug therapy for AML. Corroborative evidence for a role of MV and IGF-I signaling in shaping the leukemic microenvironment comes from the detection of human IGF-1R mRNA in cDNA prepared from mouse stromal cells (OP9) co-cultured for 48 hours with primary AML cells across a cell impermeable transwell barrier. To determine if the mRNA is transferred in a biologically significant quantity, we developed a plasmid-based quantitative PCR method to measure and compare transferred transcripts to steady state IGF-1R mRNA levels in the AML cell line HL-60. Results indicated that 0.3 HL-60 cellular equivalents of the human IGF-1R transcript were detected per OP9 cell following co-culture with primary AML cells. These data imply that transfer occurs at a level that may be functionally relevant. To further explore the potential role of MV in AML-stroma signaling, experiments are underway with HL-60 cells stably transfected with a pcDNA vector encoding the human IGF1R with a C-terminal FLAG tag. These HL-60:IGF1R-FLAG cells release MV that contain the IGF1R-FLAG transcript and will serve as a model to explore the downstream intracellular RTK signaling. In conclusion, we report for the first time the production of MV containing protein and disease-specific transcripts by AML blast cells, the selective enrichment in MV of disease-relevant transcripts, and direct cell-cell transfer of leukemia cell mRNAs to bystander cells. Our findings underscore the potential role of MV in shaping the AML microenvironment through lateral transfer of RTK mRNA transcripts. We propose a role for MV during disease progression and highlight their potential as highly sensitive, cell-free, and minimally invasive AML biomarkers. Disclosures:No relevant conflicts of interest to declare.